Vaccine

ABSTRACT

The present invention relates to compositions comprising at least one purified PorA protein antigen and at least one purified FetA protein antigen. In particular, said PorA/FetA antigens are antigenically variable antigens comprising the variable regions of PorA/FetA. Specific combinations of PorA/FetA epitopes are presented for example in Table 3. The invention also relates to methods of immunisation comprising administering said compositions, and to methods for producing compositions. Preferably the compositions are purified protein compositions. Preferably the compositions are vaccine compositions.

FIELD OF THE INVENTION

The invention relates to vaccine compositions and to their design. Inparticular the invention relates to vaccine compositions for protectionagainst N. meningitidis, including protection against N. meningitidisserogroup B.

BACKGROUND OF THE INVENTION

Neisseria meningitidis, a common commensal inhabitant of the humannasopharynx, is a major cause of bacterial meningitis and septicaemiaworldwide. Acapsulate meningococci are essentially avirulent and onlyfive of the thirteen chemically and immunologically distinctmeningococcal capsular polysaccharides, which define meningococcalserogroup, are frequently associated with invasive disease. Althoughprotein-polysaccharide conjugate vaccines offer the possibility ofprotection against meningococcal disease caused by serogroups A, C, Yand W135, this approach has not been successful for serogroup Bmeningococci. Furthermore, comprehensive prevention does not appearpossible with polysaccharide-based vaccines alone. Problems arise fromthe fact that the serogroup B polysaccharide structure is poorlyimmunogenic. Further problems arise due to its similarity to sialylatedglycopeptides on human cells.

Prior art vaccines have often made use of purification of so called“blebs” which represent vesicles shed from the cell surface of theparticular organism of interest. However, such a crude product carriesmany problems. For example, there is wide variation in the compositionof these blebs. There is no reliable way of controlling which proteinsare included or excluded from these blebs. These blebs may or may notinclude polysaccharide-coating elements of the organism of interest. Theproportions of the various components of the blebs in relation to oneanother cannot be reliably determined. The composition of these blebscannot be easily determined or controlled.

Many attempts have been made to develop vaccines based on thesub-capsular antigens, especially the outer membrane proteins (OMPs).Meningococcal OMPs are highly diverse and, although OMP-containing outermembrane vesicle (OMV) vaccines have been effective against theparticular epidemic strain from which they were made, levels ofpotentially cross-protective immune responses to heterologous strainshave been disappointing.

One prior art vaccine is the so called OMV (outer membrane vesicle)vaccine. This has included up to nine different PorA proteins. This ninefold vaccine is made up of OMVs produced from three different strains ofbacterium, each bearing its naturally occurring PorA plus two furtherPorA's which have been engineered into each of the three strains. Thisis the so called “conventional” vaccine. As such, it suffers fromsimilar problems are as present in other parts of the prior art such asare associated with vesicle vaccines generally. These problems includedifficulty in controlling the levels of antigen present, thedifficulties associated with the fact that this is a vesicle basedvaccine and include the crude nature of the preparation used.

OMV vaccines containing outer membrane proteins (OMPs) have been used inthe control of epidemics caused by single strains in Norway and Cuba.Due to the high antigenic diversity of many OMPs among differentstrains, immune responses to vaccines of this type are usually limitedto the strains used in their manufacture or their close relatives.Consequently, this approach does not provide effective control ofendemic serogroup B disease, which is attributed to diverse strains.

Feavers et al 1996 (Clinical and Diagnostic Laboratory Immunology Volume3 pp 444-450) discusses the antigenic diversity of the meningococcalPorA outer membrane protein. A wide ranging serological and nucleic acidtyping study is described. Medical problems presented by antigenicvariability are discussed in relation to vaccine design. Although PorAprotein vaccines are mentioned, they are mentioned in the context ofillustrating the problems associated with the design of proteincomponent vaccines directed against a variable antigen. Indeed, theunpredictability of these vaccines is discussed, and the efforts towardsidentification of conserved antigens are summarised.

Thompson et al 2003 (Microbiology Volume 149 pp. 1849-1858) reportsextensively on the antigenic diversity of the meningococcal FetAprotein. FetA is shown to be very highly diverse. Out of 107 individualN. meningitidis isolates examined, 60 different FetA alleles wereidentified. These 60 alleles encoded 56 different FetA proteinsequences. Thompson et al explained that the diversity of this FetAprotein will compromise its effectiveness as a vaccine component.Indeed, it is thought to be present in certain outer membrane vesicle(OMV) vaccines, but these vaccines suffer from the problem of strainspecificity. Indeed, in order to expand the coverage of protection usingOMV vaccines, OMVs derived from each invasive genotype predominant in aparticular area would be needed to be included in vaccine formulations.The conclusions reached in Thompson et al are that FetA is so diverse itis likely to be largely ineffective in vaccine formulations and thatconserved antigens probably offer the best way forward in vaccinedevelopment.

The present invention seeks to overcome problem(s) associated with theprior art.

SUMMARY OF THE INVENTION

The present invention is based on the surprising finding thatantigenically variable antigens can be exploited in vaccinating toprovide broad protection against a range of different strains of thetarget organism. Previously, antigenically variable antigens wereconsidered to be a very poor candidate component for vaccinecompositions due to their variability and various other difficultieswhich have been outlined above. Variable antigens have previously onlybeen considered suitable for strain-specific vaccination. However, asexplained in detail herein, the deep analysis of the phylogeny of thesevarious variable antigens has surprisingly revealed that certaincombinations of said antigens may be exploited in combination to providean effective vaccine composition.

In particular, in the field of meningococcal vaccines, the combinationof PorA and FetA antigens is found to be particularly advantageous dueto the very similar immune responses elicited by each antigenindividually. These responses are not only similar in nature but alsosimilar in strength. These advantageous properties allow immunodominanceeffects to be easily removed from vaccine compositions as describedherein.

Furthermore, and most importantly, the invention is based on deeperinsights into the representation of PorA and FetA antigens across theextremely broad range of antigenically diverse meningococcal isolateswhich have been studied. It is surprisingly shown that selection of arelatively small number of variable antigens from PorA and from FetAcombined, can provide protection against an extremely broad range ofthose disease associated isolates.

Thus in a first aspect the invention relates to a composition comprisingat least one purified PorA protein antigen and at least one purifiedFetA protein antigen. It is surprisingly shown herein that theantigenically variable regions of these proteins can be used incompositions, preferably vaccine compositions, providing broadprotective coverage. Preferably the PorA/FetA antigens are antigenicallyvariable antigens comprising the variable regions of PorA/FetA.

The variable regions of PorA/FetA are characterised and epitopes havebeen described within those regions. As is discussed in more detailbelow, combinations of those variable epitopes can be exploited toprovide effective vaccine compositions despite the variability of theepitopes. The epitopes are listed in Table 3. Preferably the compositioncomprises at least one PorA VR1 epitope and at least one PorA VR2epitope, wherein said epitopes are selected from the list presented inTable 3. Preferably the composition comprises at least one FetA epitopeselected from the list presented in Table 3.

Certain defined combinations of epitopes advantageously provideprotection against specifc groups of disease causing isolates. Oftenthese combinations/groupings will be determined by the operator. Someparticularly advantageous combinations are presented below.

In one aspect the invention relates to a composition as described abovewherein the composition comprises PorA epitopes P1.5-2, P1.10, P1.7 andP1.13-1 and FetA epitopes F5-1 and F1-5. This provides a coreprotection. Preferably the composition further comprises PorA epitopesP1-20 and P1.9, and FetA epitope F3-1. This is especially useful inprotection against African/Asian isolates.

In one aspect the invention relates to a composition wherein thecomposition comprises PorA epitopes P1.5-1, P1.2-2, P1.5-2, P1.16, P1.5,P1.10, P1.7, P1.7-2 and P1.4, and FetA epitopes F1-5, F5-1 and F3-9.This provides an extended core protection. Preferably the compositionfurther comprises PorA epitopes P1.13-1, P1-20 and P1.9, and FetAepitopes F3-1 and F5-5. This advantageously provides a ‘standard’vaccine having more than 88% coverage of global isolates. Preferably thecomposition further comprises PorA epitopes P1.19 and P1.15. Thisadvantageously provides an ‘enhanced’ vaccine having more than 90%coverage of global isolates.

In another aspect, the invention relates to an extended core compositionas described above wherein the composition further comprises PorAepitopes P1.2, P1.19 and P1.15, and FetA epitope F1-7. Thisadvantageously provides a composition, preferably a vaccine composition,directed to protection against European/USA isolates.

In another aspect, the invention relates to a composition as describedabove wherein the composition comprises PorA epitopes P1.5 and P1.2 andFetA epitopes F3-6 and F5-1. This advantageously provides protectiondirected at the ST-11 complex isolates.

In another aspect, the invention relates to a composition as describedabove wherein the composition comprises PorA epitopes P1.7, P1.16, P1.19and P1.15 and FetA epitope F3-1. This advantageously provides protectiondirected at the ST-32 complex isolates.

In another aspect, the invention relates to a composition as describedabove wherein the composition comprises PorA epitopes P1.7-2 and P1.4and FetA epitopes F1-5 and F1-7. This advantageously provides protectiondirected at the ST-41/44 complex isolates.

Additional components may be used to advantageously included in thecomposition(s) in order to supplement the immune responses generated.Preferably the compositions described above further comprise one or morecomponents selected from the group consisting of transferrin bindingproteins, PorB, Opa, NspA. Preferably said further component is selectedfrom the group consisting of transferrin binding protein, PorB and Opa.Preferably said further component is Opa.

Preferably the composition is a purified protein composition.

Preferably the composition is a vaccine composition.

In another aspect, the invention relates to methods of immunising asubject against Neisseria meningitidis infection comprisingadministering to said subject an effective amount of a composition,preferably a vaccine composition, as described above.

In another aspect, the invention relates to methods of inducing animmune response against Neisseria meningitidis in a subject comprisingadministering to said subject an effective amount of a composition,preferably a vaccine composition, as described above.

In another aspect, the invention relates to a method of producing avaccine composition against an organism comprising the steps of

-   -   (i) providing surface protein sequences for said organism;    -   (ii) selecting from said surface proteins those which are        antigenically variable;    -   (iii) determining the incidence of each of said antigenically        variable proteins in clinical occurrences of said organism;    -   (iv) selecting a sub-group of antigens from said antigenically        variable surface proteins to provide optimal representation of        different isolate(s) of the organism whilst including the        minimum number of individual antigens; and    -   (v) providing the antigens selected in step four in a vaccine        composition.

The compositions may advantageously be balanced to avoid immunodominanceeffects. Thus, preferably the individual antigens for inclusion in thecomposition are further selected as inducing equivalent immuneresponses. The compositions may advantageously be produced to cover asmany disease related strains as possible. Thus, preferably said antigensare further selected to be representative of variation between multipledisease associated strains of said organism.

In one embodiment, the number of antigens included in a composition maybe limited or predetermined. In this embodiment, step (iv) of the abovemethod would simply be adapted to selecting a sub-group of antigens fromsaid antigenically variable surface proteins to provide maximalrepresentation of different isolate(s) of the organism whilst includingthe predetermined number of individual antigens.

In another embodiment, the coverage of the composition may bepredetermined for example to a geographical spread or to a particularclone or collection of isolates. In this embodiment, step (iv) of theabove method would simply be adapted to selecting a sub-group ofantigens from said antigenically variable surface proteins to providethe desired coverage by choosing a single antigen providing the greatestpossible increase in coverage and adding this to the sub-group ofselected antigens until addition of a further antigen would not furtherincrease the coverage of isolate(s) of the organism.

In some embodiments, a so-called ‘multiple hit’ approach may be used. A‘single hit’ approach is where at least one antigen present on eachcovered isolate is included in the composition. A multiple hit approachis where multiple antigens present on each covered isolate are includedin the composition, such as a ‘double-hit’ approach where at least twoantigens per isolate are included. In general, multiple hit approachesare preferred.

In another aspect the invention relates to a composition comprising acombination of PorA and FetA epitopes as set out in Table 3. Preferablysaid composition comprises a combination of PorA and FetA epitopes asdescribed above. Preferably said composition is a vaccine composition.Preferably said composition is an outer membrane vesicle vaccine.

Preferably compositions according to the present invention areessentially free from cellular components such as polysaccharide capsulematerial and/or vesicles. This provides the advantage that thecompositions are free from additional antigens which are not intended tobe present but can be carried by cellular debris. Furthermore, itfacilitates the regulatory approval process since the compositionsaccording to the present invention preferably do not contain cellularfractions which can be less predictable in their composition andtherefore give rise to problems of consistency and batch variation,which problems are advantageously avoided by using purified protein onlycompositions as described herein.

DETAILED DESCRIPTION OF THE INVENTION Meningococcal Diversity

In addition to antigenic diversity, meningococcal populations aregenetically highly diverse; many genotypes have been identified by theexamination of housekeeping genes that are subject to stabilisingselection for conservation of metabolic function. These genotypes havebeen identified as electrophoretic types (ETs) by multi-locus enzymeelectrophoresis (MLEE) and more recently as sequence types (STs) bymulti-locus sequence typing (MLST).

Population studies that exploit MLEE and MLST have identified equivalentgroups of related genotypes referred to as ‘clonal complexes’; these arenow named after a predominant, or central, ST, e.g. ST-1 complex, andinclude all isolates that share identical alleles with this ST at fouror more MLST loci. Isolate collections corresponding to populations ofasymptomatically carried meningococci show greatest genetic diversitywith less diversity present in collections of disease-associatedmeningococci. The latter are dominated by meningococci belonging to alimited number of clonal complexes known as the hyper-invasive lineages.

OMP Diversity

It is well established that meningococcal OMPs are highly diverse inpeptide sequence and that their genes are subject to strong positiveselection in regions encoding those parts of the proteins exposed toimmune attack. The strength of positive selection recorded in the porBgene, for example, exceeds that reported for the envelope protein ofHIV-1. These observations raise the possibility of poor efficacy and therapid spread of escape variants following the introduction of vaccinesthat include meningococcal OMPs as immunodominant constituents. Thereis, however, some evidence for structure in the extent of antigenicdiversity present in meningococcal populations and, if understood andexploited, these limitations could simplify vaccine design andimplementation.

Associations of particular serotypes (PorB variants) and serosubtypes(PorA variants) with clonal complex have been identified in severalstudies, and variants observed in PorA VR1 and VR2 are structured intonon-overlapping combinations. Both of these associations appear to bemaintained in the face of diversifying selection and high rates ofrecombination that both reassort genes among clonal complexes andgenerate mosaic genes encoding novel antigen variants. In addition, astudy of antigenic variants of the OMP TbpB in the ST-5 complexindicated that whilst antigenic variants emerged during epidemic spread,they were less fit than the parent genotype and lost during subsequenttransmission.

PorA/FetA

PorA and FetA are outer membrane proteins of N. meningitidis. Theirsequence composition is well known to a person skilled in the art. Thevariable regions of these proteins have been mapped and are described inthe literature. For convenience, reference is made to “neisseria.org”which is a central resource in which sequence information is availablefor both FetA and PorA. Each variable region comprises at least oneepitope. The terms ‘variable region’ and ‘epitope’ are sometimes usedinterchangeably herein as will be apparent from the context. This isexplained in more detail below.

The variable region sequences (i.e. epitope sequences) of both proteinsare classified according the accepted scheme known in the art and thecommon numerical designations for the different sequence variants areused throughout this document (see for example Russell et al 2004Emerging Infectious Diseases vol. 10 p 674).

Preferably the term ‘antigenically variable antigen’ refers to anantigen comprised by variable region(s) (‘VR’) of the particular proteinconcerned.

FetA has a single variable region (epitope sequence). The sequences ofall known variants of this variable region are known in the art. Forconvenience, reference is made to the list of FetA VR sequences athttp://neisseria.org/nm/typing/feta/vr.shtml.

PorA has two variable regions (epitope sequences), VR1 and VR2. Thesequences of all known variants of these two variable regions are knownin the art. For convenience, reference is made to the list of PorA VR1sequences at http://neisseria.org/nm/typing/pora/vrl.shtml, and the listof PorA VR2 sequences at http://neisseria.org/nm/typing/porahr2.shtml.PorA antigens described herein preferably comprise one VR1 and one VR2per PorA protein. Many VR1/VR2 sequence combinations are known innature, and those combinations which are not known in nature may beeasily constructed by a person skilled in the art using elementaryrecombinant molecular biology techniques to put the relevant VR1 and VR2sequences together in a PorA context as required. Where PorA epitopecompositions are given as VR1, VR2, preferably these two epitopes areprovided on a single PorA protein. However, where more than one PorAprotein is comprised by a particular composition, then the list ofepitopes for that particular composition is more important than theparticular pairings on any one PorA molecule. For example, if P1.5-1,2-2, P1.5-2,16 is specified, this will include combinations of PorAmolecules such as (P1.5-1, 2-2 and P1.5-2,16) as well as combinationssuch as (P1.5-1,16 and P1.5-2, 2-2) or supersets which include each ofthe listed epitopes. Preferably the PorA epitope combinations areprovided as written i.e. if P1.5-1, 2-2, P1.5-2,16 is specified thenpreferably the PorA is provided as (P1.5-1, 2-2 and P1.5-2,16).

Preferably the compositions only comprise the specified PorA/FetAepitopes.

PorA and FetA are protective and generate bactericidal responses in bothhumans and animal models of infection and protection. In the prior art,PorA has been a major constituent of strain-specific vaccines includedin vaccine trials or in development. While FetA has been included inprior art strain specific vaccines and is protective, it is relativelyless exploited as it is not expressed well in vitro. The PorA VRs,especially VR2, and the FetA VR are similar in that they are relativelylong surface-exposed peptides (VR2 ranges from 8-24 amino acids long;FetA VR ranges from 20-42 amino acids long) that are easily defined.

Antigen Combinations

Preferably antigens are chosen to provide similar overall immuneresponses.

For example antigens providing similar immunogenic properties areparticularly suitable for combination. Proteins having well definedvariable regions are especially suitable for use in the presentinvention. Particularly good combinations of antigens are found when theindividual antigens produce very similar immune responses. Especiallyadvantageous are antigens having a similar strength of immune responsesince this helps to remove immunodominant effects. Antigens inducing asimilar quality of immune response are also good candidates forcombination. For example, if each of the antigens elicits an immuneresponse with known bactericidal activity then this is againadvantageous. If this bactericidal activity is found at similar levelsfor the different antigens, then they are even better suited tocombination. In summary, the closer the match between immune responses(in terms of the quality and/or quantity) generated by two differentantigens, the better candidates they are for combination.

The FetA-PorA combination is particularly advantageous since the PorAvariable regions are more similar in their immunogenic properties to theFetA immunogenic regions than to other outer membrane proteinsconsidered.

Similar criteria can be applied to a selection of particularlyadvantageous third or further components of vaccine compositionsaccording to the present invention. This is discussed in more detailherein.

Further advantages of the PorA-FetA combination include theexceptionally broad coverage which it is possible to achieve withinclusion of the minimal number of individual protein antigens.

Compositions

It is disclosed herein that a combination of PorA and FetA variants in acomposition, preferably a vaccine composition, can be particularlyeffective given the strong structuring of bacterial populationsexpressing immunogenic variants of these two antigens. The survey of 78isolates (see examples) showed that as few as 6 PorA variants (P1.5-1,2-2, P1.5-2,16, P1.5,10, P1,7,13-1, P1.7-2,4, P120,9) combined with 5FetA variants (F1-5, F3-1, F5-1, F3-9, F5-5) would provide homologousprotection against all 78 isolates. This combination of PorA and FetAantigenic variants according to the present invention protects against95 of the 107 (89%) diverse meningococcal isolates used to develop MLSTfrom which the 78 representative of the hyper-invasive lineages werederived.

Form of the Antigens

Antigens may be used in any suitable form such as purified protein ornucleic acid encoding the antigens. The antigens are preferably used inthe form of purified protein antigens. The term ‘purified’ in thiscontext means essentially free from cellular components such aspolysaccharide capsule material and/or vesicles. Preferably the antigensare used in the form of essentially homogeneous protein preparations asjudged by coomassie stained SDS-PAGE.

Clearly, a purified preparation of one antigen mixed with a purifiedpreparation of another antigen to produce a composition, preferably avaccine composition, gives rise to a mixture of at least two polypeptidespecies which mixture itself will not be homogeneous since it willcomprise at least two antigen species. Thus, a composition comprisingpurified X and purified Y will be understood to be ‘purified’ in thesense explained above i.e. that it will be essentially free fromcellular components such as polysaccharide capsule material and/orvesicles. The mere presence in the overall composition of differentindividual elements X and Y does not mean that those elements are nolonger ‘purified’ by virtue only of having been combined as described.

Preferably the antigens are produced by recombinant means. Preferablythe antigens are produced in the absence of N. meningitidis, such asproduction by recombinant means in a non-N. meningitidis cell.Preferably the antigens are produced via expression in E. coli.

The production of the individual purified antigens is within the abilityof the person skilled in the art. These can be produced by any suitablemeans known in the art, for example by recombinant expression,purification and refolding (if necessary) in vitro such as described inIdanpaan-Heikkila, I., Wahlstrom, E., Muttilainen, S., Nurminen, M.,Kayhty, H., Saryas, M., and Makela, P. H. (1996) Immunization withmeningococcal class 1 outer membrane protein produced in Bacillussubtilis and reconstituted in the presence of Zwittergent or TritonX-100 Vaccine 14: 886-891; Jansen, C., Kuipers, B., van der, B. J., deCock, H., van der, L. P., and Tommassen, J. (2000) Immunogenicity of invitro folded outer membrane protein PorA of Neisseria meningitidis FEMSImmunol. Med. Microbiol. 27: 227-233; Jansen, C., Wiese, A., Reubsaet,L., Dekker, N., de Cock, H., Seydel, U., and Tommassen, J. (2000)Biochemical and biophysical characterization of in vitro folded outermembrane porin PorA of neisseria meningitidis [In Process Citation]Biochim. Biophys. Acta 1464: 284-298; Qi, H. L., Tai, J. Y., and Blake,M. S. (1994) Expression of Large Amounts of Neisserial Porin Proteins inEscherichia coli and Refolding of the Proteins into Native Trimers.Infection and Immunity 62: 2432-2439.

Antigens may be concatenated i.e. physically joined for example byproduction of multiple antigens by expression as a continuouspolypeptide chain. In one embodiment, this may be accomplished usinggenetically engineered hybrids of PorA and FetA expressing epitopes fromboth proteins. For example, the semi-variable cell-surface loop five ofPorA may be replaced with the VR from FetA, such that a PorA-FetA fusionprotein is produced comprising not only the PorA VR1 and VR2 but alsothe FetA VR. The choice of epitopes for this type of concatenation orfusion protein follows the details described herein according to thedesired protection sought. This approach advantageously reduces thenumber of physical proteins required to provide the same level ofcross-protection:

In any case, concatenation of epitopes may be carried out for simpleconvenience/optimisation of the production process, or for other reasonssuch as balancing of the induced immune response. Preferably onlyantigens inducing similar strength responses are concatenated ontosingle polypeptides. Preferably concatenation is only performed forantigens occurring within a particular protein. Preferably concatenationis avoided. Preferably individual purified antigens are prepared andstored separately until needed to produce a composition according to thepresent invention.

The precise amounts of individual antigens in partcular vaccinecompositions will typically be determined by the person working theinvention. Preferably equimolar amounts of individual antigens are used.More preferably the amounts used are balanced with regard to thestrength of immune response induced against a particular antigenspecies. Thus, if an antigen elicits a response at only half thestrength of a reference antigen, then twice the molar amount of thatantigen should be used. Similarly, if an antigen elicits a response attwice the strength of the reference antigen, then approximately half themolar amount should be used. Preferably the optimal relative proportionsand dosage levels are determined by clinicians/clinical studies.

This balancing advantageously helps to avoid immunodominance effectsproduced by inequalities between the individual antigen components ofthe vaccine compositions. This process can be seen as a simple processof titration towards an end-point of even response against each of theantigens in a given vaccine composition, preferably of even protectionagainst each of the antigens in a given vaccine composition. Thetitration is preferably performed by a serum bactericidal antibody assayor an ELISA.

Optimisation

Clearly, the best vaccine may well be the vaccine having broadestcoverage. However, to obtain the broadest coverage may require theinclusion of the greatest number of antigens into the vaccinecomposition. Therefore, preferably a balance is struck betweenminimising the number of antigens included in the composition andmaximising the coverage of protection which might be afforded by saidcomposition. These are the factors which should govern the choice ofantigens in the present invention.

In more detail, the first antigen to be chosen would be the singleantigen which occurred in the greatest number of individual isolates ofthe organism of interest. This single antigen would provide the greatestcoverage across those isolates. When considering what to choose as asecond antigen, attention should be paid to those isolates which are notyet represented by inclusion of the first antigen. In this manner, thesecond antigen should be chosen to provide the greatest coverage acrossthose so far unrepresented isolates. At this point, two antigens willhave been selected. These will have been selected to provide the bestcoverage possible for the selection of only two antigens. However, theremay still be a group of isolates which are not yet represented.Therefore, the choice of the third antigen should address those isolateswhich have not yet been represented. This iterative process of choosingand selecting antigens should be continued to achieve as high a level ofcoverage in teinis of the number of isolates covered as possible.Preferably the process should be continued until each of the knownisolates is covered.

Advantageously, if all isolates are covered yet there is still space inthe composition for inclusion of further antigens, the above process maybe continued selecting further antigens which will provide furtherimmunological responses against the maximum number of isolates. In thisway, dual responses may be generated against individual isolates. Thisis sometimes called the ‘double hit approach’. Clearly, the moreresponses which are generated against each isolate, the better chance ofproviding protective response in the host subject. Therefore, greaternumbers of antigens and greater numbers of hits against each isolate arepreferred. However, practical difficulties including considerations ofcost in preparation of the composition dictate some limitation on thenumber of antigens included in that particular vaccine composition. Thislimitation will vary from application to application. Furthermore, usingfewer antigens will almost always result in an easier to produce andultimately cheaper vaccine composition. Furthermore, there may betechnical advantages to reducing the number of vaccines in thecompositions such as elimination of immunodominance effects, ease ofbalancing the responses, and/or simplification of administration. Theactual limitation on the number of antigens included in a particularcomposition is not important to the invention. The important principleis that when choosing the antigens they are chosen according to theprocess outlined above, that is to say an iterative process in whichmaximising the coverage of protection is given the highest priority. Inthis way, whatever the actual numerical limitation on the number ofantigens included in a particular vaccine composition, a vaccinecomposition containing that limited number of antigens will alwaysprovide the greatest possible coverage when the antigens are selectedaccording to the present invention.

Thus in one aspect the number of antigens to be included in a particularcomposition will be determined before the choice of individual antigensis made. Each individual antigen is then chosen as explained above,maximising the coverage attained with the addition of each individualantigen to the composition, preferably the vaccine composition.

In another aspect, the coverage of a particular composition will bedetermined before the number of antigens to be included in thecomposition is determined. Each individual antigen is then chosen asexplained above, adding antigens one at a time until the desiredcoverage is attained.

Naturally, many compositions may involve a compromise between a desiredcoverage and a preferred limitation on the number of antigens includedin the composition. The present invention advantageously enables suchfactors to be balanced by following the guidance given herein.

In general, the fewer antigens the composition comprises, the simplerand cheaper it will be to manufacture, administer and monitor. Thereforein some aspects a low number of antigens will be advantageous.

In aspects of the invention when a composition is designed by coverage,then clearly a greater number of antigens may be desirable in order toattain that coverage and the general preference for a smaller number ofantigens will be balanced to allow the desired coverage to be attained.

The vaccine composition may comprise twenty antigens or even more.Preferably, the vaccine composition will comprise eighteen or fewerantigens, preferably sixteen or fewer antigens, preferably fourteen orfewer antigens, preferably twelve or fewer antigens, preferably elevenor fewer antigens, preferably ten or fewer antigens, preferably nine orfewer antigens, preferably eight or fewer antigens, preferably seven orfewer antigens, preferably six or fewer antigens, preferably five orfewer antigens, preferably four or fewer antigens, preferably three orfewer antigens, preferably the vaccine composition comprises twoantigens.

The definition of an antigenically variable antigen (as compared to anantigenically conserved antigen) is well known in the art for example avariable antigen is one which differs by one or more amino acids fromthe sequence of the prototype antigen. As a result of this sequencedifference it is likely to elicit an antibody response that is less than100% cross-reactive between the variant and the prototype. Variableantigens will be characterised by predominance of non-synonymous oversynonymous nucleotide substitutions.

Further Components

The compositions and/or vaccine formulations of the present inventioncomprise at least one purified PorA antigen and at least one purifiedFetA antigen. These vaccine compositions may advantageously besupplemented with further component(s) to improve the vaccines e.g. toimprove their efficacy.

This third or further component may advantageously be selected fromtransferrin binding proteins, PorB, opacity associated adhesins (Opas),NspA, N. meningitidis cell surface components such as outer membraneprotein(s) or other entity capable of eliciting or augmenting an immuneresponse. Preferably the third or further component is an outer membraneprotein. Preferably the third or further component is selected from thelist consisting of transferrin binding proteins, PorB, Opas.

Vaccine Formulations

The present invention provides a pharmaceutical/vaccine compositioncomprising a therapeutically effective amount of the PorA/FetA epitopesof the present invention and a pharmaceutically acceptable carrier,diluent or excipient (including combinations thereof).

The pharmaceutical compositions may be for human or animal usage inhuman and veterinary medicine and will typically comprise any one ormore of a pharmaceutically acceptable diluent, carrier, or excipient.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as—or in addition to—the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

Preservatives, stabilizers, dyes and even flavoring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

There may be different composition/formulation requirements dependent onthe different delivery systems. By way of example, the pharmaceuticalcomposition of the present invention may be formulated to beadministered using a mini-pump or by a mucosal route, for example, as anasal spray or aerosol for inhalation or ingestable solution, orparenterally in which the composition is formulated by an injectableform, for delivery, by, for example, an intravenous, intramuscular orsubcutaneous route. Alternatively, the formulation may be designed to beadministered by a number of routes.

Where the agent is to be administered mucosally through thegastrointestinal mucosa, it should be able to remain stable duringtransit though the gastrointestinal tract; for example, it should beresistant to proteolytic degradation, stable at acid pH and resistant tothe detergent effects of bile.

Where appropriate, the pharmaceutical compositions can be administeredby inhalation, in the form of a suppository or pessary, topically in theform of a lotion, solution, cream, ointment or dusting powder, by use ofa skin patch, orally in the form of tablets containing excipients suchas starch or lactose, or in capsules or ovules either alone or inadmixture with excipients, or in the form of elixirs, solutions orsuspensions containing flavouring or colouring agents, or they can beinjected parenterally, for example intravenously, intramuscularly orsubcutaneously. For parenteral administration, the compositions may bebest used in the form of a sterile aqueous solution which may containother substances, for example enough salts or monosaccharides to makethe solution isotonic with blood. For buccal or sublingualadministration the compositions may be administered in the form oftablets or lozenges which can be formulated in a conventional manner.

PorA/FetA protein may be prepared in situ in the subject being treated.In this respect, nucleotide sequences encoding said protein may bedelivered by use of non-viral techniques (e.g. by use of liposomes)and/or viral techniques (e.g. by use of retroviral vectors) such thatthe said protein is expressed from said nucleotide sequence.

Administration

The term “administered” includes delivery by viral or non-viraltechniques. Viral delivery mechanisms include but are not limited toadenoviral vectors, adeno-associated viral (AAV) vectors, herpes viralvectors, retroviral vectors, lentiviral vectors, and baculoviralvectors. Non-viral delivery mechanisms include lipid mediatedtransfection, liposomes, immunoliposomes, lipofectin, cationic facialamphiphiles (CFAs) and combinations thereof.

The components of the present invention may be administered alone butwill generally be administered as a composition—e.g. when the componentsare is in admixture with a suitable pharmaceutical excipient, diluent orcarrier selected with regard to the intended route of administration andstandard pharmaceutical practice.

For example, the components can be administered in the form of tablets,capsules, ovules, elixirs, solutions or suspensions, which may containflavouring or colouring agents, for immediate-, delayed-, modified-,sustained-, pulsed- or controlled-release applications.

If the administration is via a tablet, then the tablet may containexcipients such as microcrystalline cellulose, lactose, sodium citrate,calcium carbonate, dibasic calcium phosphate and glycine, disintegrantssuch as starch (preferably corn, potato or tapioca starch), sodiumstarch glycollate, croscannellose sodium and certain complex silicates,and granulation binders such as polyvinylpyrrolidone,hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC),sucrose, gelatin and acacia. Additionally, lubricating agents such asmagnesium stearate, stearic acid, glyceryl behenate and talc may beincluded.

Solid compositions of a similar type may also be employed as fillers ingelatin capsules. Preferred excipients in this regard include lactose,starch, a cellulose, milk sugar or high molecular weight polyethyleneglycols. For aqueous suspensions and/or elixirs, the agent may becombined with various sweetening or flavouring agents, colouring matteror dyes, with emulsifying and/or suspending agents and with diluentssuch as water, ethanol, propylene glycol and glycerin, and combinationsthereof.

The routes for administration (delivery) include, but are not limitedto, one or more of:

oral (e.g. as a tablet, capsule, or as an ingestable solution), topical,mucosal (e.g. as a nasal spray or aerosol for inhalation), nasal,parenteral (e.g. by an injectable form), gastrointestinal, intraspinal,intraperitoneal, intramuscular, intravenous, intrauterine, intraocular,intradermal, intracranial, intratracheal, intravaginal,intracerebroventricular, intracerebral, subcutaneous, ophthalmic(including intravitreal or intracameral), transdermal, rectal, buccal,vaginal, epidural, sublingual.

In a preferred aspect, the composition is delivered by injection.

It is to be understood that not all of the components of the compositionneed be administered by the same route. Likewise, if the compositioncomprises more than one active component, then those components may beadministered by different routes.

If a component of the present invention is administered parenterally,then examples of such administration include one or more of:intravenously, intra-arterially, intraperitoneally, intrathecally,intraventricularly, intraurethrally, intrasternally, intracranially,intramuscularly or subcutaneously administering the component; and/or byusing infusion techniques.

For parenteral administration, the component is best used in the form ofa sterile aqueous solution which may contain other substances, forexample, enough salts or glucose to make the solution isotonic withblood. The aqueous solutions should be suitably buffered (preferably toa pH of from 3 to 9), if necessary. The preparation of suitableparenteral formulations under sterile conditions is readily accomplishedby standard pharmaceutical techniques well-known to those skilled in theart.

As indicated, the component(s) of the present invention can beadministered intranasally or by inhalation and is conveniently deliveredin the form of a dry powder inhaler or an aerosol spray presentationfrom a pressurised container, pump, spray or nebuliser with the use of asuitable propellant, e.g. dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkanesuch as 1,1,1,2-tetrafluoroethane (HFA 134A™) or1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA™), carbon dioxide or othersuitable gas. In the case of a pressurised aerosol, the dosage unit maybe determined by providing a valve to deliver a metered amount. Thepressurised container, pump, spray or nebuliser may contain a solutionor suspension of the active compound, e.g. using a mixture of ethanoland the propellant as the solvent, which may additionally contain alubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, forexample, from gelatin) for use in an inhaler or insufflator may beformulated to contain a powder mix of the agent and a suitable powderbase such as lactose or starch.

The component(s) of the present invention may also be dermally ortransdermally administered, for example, by the use of a skin patch.

It will be understood that these regimes include the administration ofthe substances sequentially, simultaneously or together.

Dose Levels

Typically, a physician will determine the actual dosage which will bemost suitable for an individual subject. The specific dose level andfrequency of dosage for any particular patient may be varied and willdepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the age, body weight, general health, sex, diet, mode and timeof administration, rate of excretion, and the individual undergoingtreatment.

Depending upon the need, the agent may be administered at a dose of from0.00001 ug/Kg body weight to 5 mg/Kg body weight, preferably 0.0001ug/Kg to 5 mg/Kg, preferably 0.001 ug/Kg to 1 mg/Kg, preferably 0.01ug/Kg to 500 ug/Kg, preferably 0.02 ug/Kg to 300 ug/Kg body weight.Preferably the composition comprises up to about 25 ug of each PorA/FetAcomponent.

In a preferred embodiment, a dose of approximately 3 ug is administeredto a child of approximately 3-4 Kg in weight.

Preferably compositions according to the present invention areessentially free from cellular components such as polysaccharide capsulematerial and/or vesicles.

Preferably the vaccine compositions according to the present inventionare protein vaccine compositions.

Preferably the vaccine compositions of the present invention comprise anadjuvant. Any suitable adjuvant known in the art may be employed in thepresent invention. The person skilled in the art will vary the adjuvantand/or quantities or proportions thereof according to the immuneresponse required. Preferably this adjuvant is Aluminium hydrogel.

Specific exemplary compositions are set out herein, in particular withreference to Table 3.

The invention will now be described by way of example with reference tothe following figures:

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows phylogenetic analysis of 78 hyperinvasive meningococciusing (A)7 concatenated housekeeping gene sequences (3,284 base pairs)and (B) 3 concatenated antigen gene sequences (4,209 base pairs), Eachisolate is colour coded according to clonal complex, as defined byMultilocus Sequence Typing (MLST).

FIG. 2 shows coomassie stained PAGE protein samples.

FIG. 3 shows PAGE protein samples from various differenttreatments/conditions (see examples for details).

EXAMPLE 1 Survey and Vaccine Compositions

This example presents vaccine compositions and methods for theirproduction.

To inform the choice of variants to be included in meningococcalvaccines according to the present invention, a survey of the variationof three major OMPs, PorA, PorB, and FetA, was undertaken in acollection of 78 meningococci representing the seven hyper-invasivelineages associated with most meningococcal disease outbreaks of thelatter half of the twentieth century. Surface protein sequences for theorganism were provided. Analysis of the nucleotide sequences of thegenes encoding these antigens revealed strong evidence for positiveselection acting on those parts of the genes previously described asencoding immunogenic regions of all three proteins. Phylogeneticanalysis of concatenated antigen gene sequences generated clusters thatwere congruent with clonal complex, although this congruence was lessapparent when individual loci were analysed. Further, there was evidencefor both the persistence of particular combinations of antigen variantsduring decades of global spread and the presence of identical antigencombinations in otherwise unrelated isolates. This antigenic structuringgreatly simplified the number of components required for a vaccine thatpotentially provided cross-protection against all seven hyper-invasivelineages. For example, a vaccine according to the invention containingonly six PorA combined with five FetA variants provides protectionagainst all of the 78 isolates included in this study (see below),

Materials and Methods Growth of Meningococi and DNA Preparation.

A total of 78 meningococcal isolates, representative of the majormeningococcal disease outbreaks reported in the latter half of thetwentieth century were chosen for this analysis (Table 1). Theseisolates were previously characterised by multi-locus sequence typing(MLST), and included: 37 serogroup A meningoocci (14 ST-1 complex, 11ST-4 complex, 12 ST-5 complex); 10 isolates from ST-11 complex (8serogroup C and 2 serogroup B); 8 isolates from ST-8 complex (5serogroup B and 3 serogroup C); 10 ST-32 complex organisms (9 serogroupB, 1 serogroup C); 13 isolates from ST-41/44 complex (all serogroup B).Isolates were propagated on heated blood agar plates in an atmosphere of5% CO₂ for 8-16 hours. Approximately 10⁷-10⁹ colony forming units wereused to prepare genomic DNA using an ‘Isoquick Nucleic Acid ExtractionKit’ (Orca Research Inc.), according to the manufacturer's protocol.

Nucleotide Sequence Determination and Gene Nomenclature.

PCR amplification and nucleotide sequence determination of themeningococcal porA, porB, and fetA genes was as described previously(Suker, 1994 Mol. Micro. Vol 12 p. 253; Urwin, 1998 Epid. and Inf. Vol121p. 95; Thompson, 2003 Microbiology Vol 149 p. 1849). Nucleotidesequence data for forward and reverse strands were assembled with theSTADEN software package, reformatted into ‘GCG’ format and aligned tomaintain maximum positional homology using the SEQLAB program within theGCG software package Version 10.1 (Genetics Computer Group, Madison,Wis.). Using the Molecular Evolutionay Genetics Analysis (MEGA) softwarepackage version 2.0, pairwise comparisons were performed on each set ofaligned sequences to identify distinct alleles; allele numbers were thenassigned to each unique porA, porB, and fetA gene sequence on the basisof previously defined nomenclature systems (Thompson, 2003 ibid).

Variable region (VR) Identification.

The identification of the VRs of PorA and FetA was straightforward:nucleotide sequences encoding the defined PorA variable epitopes, VR1and VR2, were translated and identified by querying the PorA VR sequencedatabase located at http://neisseria.org/nm/typing/pora/. The amino acidsequence variants determined for the FetA VR were also identified bydatabase interrogation at http://neisseria.org/nm/typing/feta/.

PorB VR identification was more complicated, as PorB proteins often havediscontinuous epitopes, where several of the eight PorB surface-exposedvariable loops are involved in epitope formation. As only loops II andIII are essentially invariant in PorB, it was necessary to identifyamino acid sequence variation in loops I, IV-VIII when determining PorBepitope diversity. Variation in PorB was therefore determined bypairwise comparisons of the aligned amino sequences corresponding toeach surface loop, with reference to a previously published scheme(Sacchi, 1998 Clinical and Diagnostic Lab. Imm. Vol 5 p. 348). Theresults were reported using a scheme slightly modified from that ofPoolman et al. (Frasch, 1985 Reviews of Infectious Diseases Vol. 7 p.504), with the format serogroup:serotype:subtype:FetA type, thus:A:4,21:P1.5-2,10:F5-1. Thus the antigenically variable proteins wereselected.

Data Manipulation and Analysis.

Phylogenetic trees were constructed using the maximum likelihood (ML)method available in the PAUP* package. The GTR model of nucleotidesubstitution was used, with values for the nucleotide substitutionmatrix, the proportion of invariant sites, and the shape parameter (+)of a gamma distribution of rate variation among sites (with 4categories) estimated during tree reconstruction. ML phylogenies wereconstructed using concatenated DNA sequences for (i) the 7 housekeepinggene fragments employed in MLST, giving a total sequence length of 3,284bp and (ii) the three antigen gene sequences giving a total length of4,209 base pairs.

Results

The incidence of each of the antigenically variable proteins in clinicaloccurrences was determined as follows.

Diversity of OMP Genes and Proteins.

The lengths of the nucleotide sequences when aligned for analysis were:fetA, 2031 bp; porA, 1155 bp; and porB, 1023 bp. Similar levels ofsequence diversity were observed at the three loci (Table 2) with 33porA alleles encoding 33 PorA sequences, 33 fetA genes encoding 31 FetAsequences, and 31 porB alleles encoding 28 PorB sequences. Two distinctallele classes were present at the porB locus, porB2 and porB3, and themajority of differences among porB alleles were due to differencebetween these classes; there was less diversity within porB2 and porB3alleles and their encoded proteins when analysed separately. Peptidesequence variation was identified in regions of the proteins implicatedin immune responses in animals and man. At the FetA VR, 16 uniquepeptide sequences were identified. There were 12 PorA VR1 and 18 PorAVR2 peptides sequences (26 unique VR1, VR2 combinations), and 20 uniquecombinations of the peptide sequences corresponding to loops I, IV-VIIIof the PorB protein (Table 1, Table 2).

Comparison of Phylogenies Obtained from Antigen Genes and HousekeepingGenes.

A ML tree constructed with concatenated housekeeping genes clustered theisolates into their clonal complexes (FIG. 1A) with the exception of theST-8 complex isolate B6116/77 which possessed a pdhC allele that wasdivergent from those present among other ST-8 isolates. Similar clustersof isolates were observed in the ML tree constructed from theconcatenated sequences of the three antigen genes, although the deeperbranching patterns of the two trees were different (FIG. 1B). Theisolates belonging to the ST-1, ST-4, and ST-5 complexes, which weremostly serogroup A, formed a Glade in the housekeeping gene tree but notin the antigen gene tree. Conversely, the isolates belonging to the ST-8and ST-11 complexes formed a Glade in the antigen gene tree, as a resultof these isolates possessing porB alleles belonging to the porB2 alleleclass, whilst the remaining isolates possessed porB alleles of the porB3class. The Glade comprising isolates of the ST-4 complex and ST-41/44complex in the antigen gene tree was a consequence of isolates sharingclosely related fetA and porA genes. The clustering of two ST-32 complexisolates (204/92 and BZ83) with ST-1 isolates reflected identity orsimilarity of fetA, porA and porB alleles with ST-1 complex organisms.The oldest isolate in the collection (A4/M1027, ST-4) isolated in 1937in the USA fell outside the otherwise closely related cluster ofsequences formed by ST-4 complex meningococci because it had a porA-16allele at the porA locus (Table 1), an allele predominant among ST-1complex organisms. Three ST-1 complex meningococci did not cluster withthe other ST-1 isolates due to fetA sequence diversity, isolates 20 and254 having a fetA-8 allele and isolate 129 a fetA-57 allele, rather thanthe fetA-3 allele observed in most ST-1 complex isolates. Two ST-41/44complex isolates (NG E30 and NG H36) did not cluster with the remainingisolates belonging to this complex as a result of divergent fetA andporA alleles.

Distribution of FetA and PorA Antigenic Variants Among Clonal Complexes.

There was similarity in patterns of variation identified in FetA andPorA protein sequences. Most variation was restricted to surface exposedloops 1 and 4 in PorA, corresponding to VR1 and VR2, and was foundmainly in one surface exposed loop in FetA, corresponding to the FetAVR. Furthermore, variants of the major immunogenic regions of both FetAand PorA proteins were unevenly distributed among clonal complexes, withparticular combinations of the three antigenic regions associated withindividual clonal complexes (Table 1). The most common combination amongthe ST-1 complex isolates was P1.5-2,10;F5-1 (6/10 isolates) while ST-4complex isolates were predominantly (6/10) P1,7,13-1:F1-5 and ST-5complex isolates were predominantly (9/12) P1.20,9:F3-1. The majority(9/13) of ST-41/44 isolates were P1.7-2,4:F1-5. The ST-8 complexisolates analysed here contained a number of variants with nopredominant VR sequences, although there was a predominance of the P1.5VR1 sequence and its variants P1.5-1 and P1.5-2, the VR2P1.2 and itsP1.2-2 variant and the FetA VR F3-9. In some cases, e.g. isolates 255(ST-4 complex), S4355 (ST-5 complex) and 400 (ST-41/44 complex),departure from the commonest combination was at a single variableregion; in other cases, multiple differences were present. Among ST-11complex isolates, for example, were two combinations with distinctpeptide sequences: four isolates were P1.5,2:F1-1 and threeP1,5,2-1:F5-5. Variants of the P1.7,16:F3-3 combination dominated theST-32 complex organisms. In two cases, isolates with unrelated STsexhibited identical PorA and FetA types: the ST-41/44 complex isolate NGE30 was identical to ST-8 complex isolate BZ163, both beingP1.21,16:F1-7, although these isolates did not share identical porA andfetA alleles. One of the ST-32 isolates was identical to the majorityantigenic type exhibited by ST-1 complex organisms, P1.5-2,10:F5-1although again the allele sequences were not identical.

Distribution of PorB Antigenic Variants Among Clonal Complexes.

Amino acid variation was identified in all surface exposed loops of PorBexcept loops II and III. Conservation of PorB sequences was observed inall clonal complexes; extensive evidence of mosaic gene structureindicated that it was likely that most of this diversity had arisen byreassortment of known sequences. The majority of ST-1 complex organisms(11/14) possessed PorB3 proteins that were identical in all surfaceloops and were phenotypically identified as serotype 4,21. The PorB3proteins of isolates 393 and 322/85 differed from the serotype 4,21sequence only in variable loop I, while isolate 79126 expressed aserotype 4 PorB3 protein which was divergent in variable loops I,VI-VIII. All ST-4 complex meningococci possessed serotype 4,21 PorBproteins, although minor variation in surface loop VIII was identifiedin one isolate (2059001). The serotype 4,21 PorB3 protein identifiedamong ST-4 complex organisms was distinguished from that identified inST-1 complex and ST-5 complex meningococci by two amino acid changes invariable loop IV. Seven of ten ST-5 complex isolates had serotype 4,21PorB3 proteins identical to those seen among ST-1 complex, while theremaining three isolates (92001, 11-004 and 80049) had one or twovariant loop sequences in PorB3. Most ST-8 complex isolates (5/8) hadidentical serotype 2b PorB2 amino acid sequences, with amino acidchanges observed in either loop IV or loop VIII of the remaining threeisolates, 94/155, BZ10 and AK22 (BZ10 still reacted with the serotype 2bmonoclonal antibody despite this variation). The PorB protein was wellconserved among ST-11 complex meningococci: nine of ten isolatespossessed identical PorB2 variable loops and corresponded to serotype2a; isolate 90/18311 had variant amino acid sequences in loops V-VII andwas not serotypable. Seven of ten ST-32 complex isolates examined hadidentical PorB3 proteins corresponding to serotype 15, while divergentsequences associated with reactivity with serotype 4 mAbs wereidentified in isolates 204/92 and BZ83. The remaining isolate (BZ169)possessed a PorB3 amino acid sequence identical to a serotype 1reference strain. Among ST-41/44 complex organisms, four isolates sharedidentical PorB3 amino acid sequences corresponding to serotype 4. Threeisolates (88/03415, NG E30, AK50) had variant PorB sequences, but werestill defined as serotype 4 as they possessed the loop VI amino acidsequence recognised by serotype 4 monoclonal antibodies. Three isolates(NG H15, NG H36, N31905) had variant PorB3 sequences corresponding toserotype 8, while the remaining three ST-41/44 complex organismspossessed variant PorB3 proteins composed of novel combinations ofvariable loop sequences that were not typable by serological methods.

Temporal and Geographic Distribution of Antigen Gene Variants andCombinations.

A number of antigen gene alleles exhibited wide temporal andgeographical distributions. The porA-16 allele was identified in 16meningococci isolated in 15 countries from five continents over a periodof 55 years; porB3-26 was identified in 10 isolates obtained from 9countries including India, the USA, and several African countries over aperiod of 53 years. The most widely distributed fetA allele in thiscollection was fetA-5 which was present in 11 isolates originating from9 countries, mainly from Africa but including China and India, over 27years. A number of sequences encoding the immunogenic variable regionsof these proteins also exhibited longevity and global distribution; theFetA VR variant F1-5 was present in 26 members of the isolate collectionincluding the oldest (A4/M1027, isolated in the USA in 1937) and themost recent isolate examined, (N45/96, isolated in Norway in 1996)(Table 1).

Discussion

The present analysis has, for the first time, assessed the allelediversity present at three distinct OMP-encoding loci in the majorhyper-invasive meningococcal lineages. The results confirmed theextensive diversity of these proteins, with between 28 and 33 peptidesequences observed for each protein and provided evidence for thestructuring of combinations of these variants.

Although recombination has erased most of the deeper phylogenetic signalin meningococcal populations, groups of related genotypes, likely tocomprise meningococci that share a common ancestor, can be identified bya number of means. MLST resolves these related genotypes as clonalcomplexes, which are defined by allelic differences rather thannucleotide sequence comparisons per se, to account for frequentrecombination. The ML trees for both housekeeping gene sequences andantigen gene sequences generated clades that were largely congruent withclonal complex for the representatives of the seven hyper-invasivelineages analysed here. While congruence of phylogenies based onhousekeeping genes with clonal complex designation was unremarkable, thedistribution of antigen gene sequences illustrated in the phylogeneticanalysis was unexpected.

Strictly clonal processes cannot account for congruence of thecombinations of antigen variants with clonal complex as themeningococcus experiences frequent recombination. The predictions ofclonality are further violated by the presence of identical antigen genesequences throughout the data set, regardless of clonal complex. The‘epidemic clone’ concept, in which non-clonal populations can betransiently dominated by particular genotypes that share a recent commonancestor, could be invoked to explain the observations. However, thiswould not explain the longevity of antigen combinations over decades ofglobal spread nor the two examples of the presence of identical antigenvariant combinations in otherwise genetically unrelated isolates.

The ‘epidemic clone’ population structure also provides no explanationfor the non-overlapping nature of the antigen combinations observed Inthe majority of cases, the clonal complexes representative of particularhyper-invasive lineages have predominant antigen gene combinations thatshare no major antigen variants with other clonal complexes. There weresome examples of sharing identical antigen variants among combinations,for example, the ST-4 complex shared the P1.7 variant with ST-32 complexand the F1-5 variant with ST-41/44 complex; however, neither of thelatter clonal complexes have been recently reported in Africa, where allof the recent ST-4 isolates originated, therefore it is possible thatthese overlapping antigen variant complexes are not presentsimultaneously in the same transmission system. By contrast, althoughpossibly related by descent, all of the serogroup A-associated clonalcomplexes that have recently caused disease in Africa (ST-1, ST-4 andST-5) have distinct, non-overlapping antigen gene combinations.

Models of strain structuring on the basis of host immunity provide atheoretical framework that can accommodate the observed combinations ofantigenic variants. These models postulate that, within a giventransmission system, pathogen strains that share immunological variantsare disadvantaged. In this respect, it was noteworthy that a number ofclonal complexes contained antigenic variant combinations that werealtered at multiple antigens. There was evidence for twoglobally-circulating strains within the ST-11 complex, P1.5,2:F1-1 andP1,5,2-1:F5-5 and one of these variants was associated with twodifferent capsules, with two isolates that were B:2a:P1.5,2:F1-1 and twoC:2a:P1.5,2:F1-1. At least two ST-32 complex variants spread widely fromthe mid-1970s onwards and these also displayed non-overlappingcombinations of OMP antigen variants. The ‘Norwegian’ strain wastypically B:15:P1.7,16;F3-3 while the ‘Spanish’ strain wasB:4:P1.19,15;F5-1. A further variant identified in the Netherlands andthe UK was notable for being identical at the level of OMPs(4:P1.5-2,10:F5-1) with many ST-1 complex isolates. A final variantidentified in Chile, whilst bearing some similarities with the Norwegianstrain, also differed at many OMPs being B:15:1.7,3:F3-1. Many examplesof these strains within the ST-32 complex have been described previouslyby serological and molecular technique. Many of the exceptions to thenon-overlapping structure involved PorB and PorA VR1; there is someevidence that these antigens may be relatively less immunogenic thanPorA VR2. In conclusion, it is possible that meningococcal strains ortransmission variants may, at least in part, be defined by OMP variantcombinations, especially those of FetA VR and PorA VR2. Thus, asub-group of antigens was selected from the antigenically variableproteins found in clinical occurrences. This selection is extended tothe epitope level as explained.

Whatever the mechanism by which meningococcal antigen variants arestructured, the present invention makes use of the observed structuringin novel vaccine design.

The results of this survey are consistent with a limited repertoire ofantigen variant combinations dominating the major meningococcalhyper-invasive lineages. The non-overlapping nature of the combinationsobserved was consistent with strain structure imposed by herd immunity.If this is the case, some low frequency variants in the population mayhave a short term selective advantage within a single host, but there issome evidence that such variants are likely to be less fit duringepidemic spread. This framework further predicts that novel variants canemerge and spread from time to time if they are distinct at multipleloci encoding immunodominant antigen variants, and that distinctgenotypes can possess identical antigen types; both of these phenomenaare present in this data set. Moreover, as bacteria are less likely tochange, by mutation or recombination, at two distantly located genessimultaneously, a vaccine that targets at least two distinct variableantigens will exhibit improved efficacy over a vaccine with multiplevariants of a single antigen.

The isolates employed in this analysis represent the majorhyper-invasive lineages reported over the last 60 years. Thus theinvention provides relatively simple meningococcal OMP-based vaccineseffective against all hyper-invasive lineages, regardless of serogroup.

Indeed, the survey showed that as few as 6 PorA variants (P1.5-1, 2-2,P1.5-2,16, P1.5,10, P1,7,13-1, P1.7-2,4, P120,9) combined with 5 FetAvariants (F1-5, F3-1, F5-1, F3-9, F5-5) provides homologous protectionagainst all 78 isolates. This combination of PorA and FetA antigensaccording to the present invention variants protects against 95 of the107 (89%) diverse meningococcal isolates used to develop MLST from whichthe 78 representative of the hyper-invasive lineages were derived. Thusthe invention provides a vaccine composition comprising 11 differentantigens, wherein said 11 antigens comprise six PorA antigens and fiveFetA antigens. In more detail, the invention provides a vaccinecomposition comprising 6 PorA variants (P1.5-1, 2-2, P1.5-2,16, P1.5,10,P1,7,13-1, P1.7-2,4, P120,9) combined with 5 FetA variants (F1-5, F3-1,F5-1, F3-9, F5-5).

TABLE 1 The porA, fetA and porB allele sequences of 78 hyper-invasivemeningococci. Sero type^(b) Allele Epitope^(a) (Por Clonal complexIsolate Year Country Serogroup ST porA fetA porB PorA_VR1 PorA_VR2FetA_VR B) ST-1 complex/subgroup I/II 6748 1971 Canada A 1 17 3 3-6018-1  3 F5-1  4.21 20 1963 Niger A 1 16 8 3-60 5-2 10 F1-7  4.21 2541966 Djibouti A 1 16 8 3-60 5-2 10 F1-7  4.21 129 1964 West Germany A 116 57 3-60 5-2 10 F3-6  4.21 371 1980 India A 1 16 30 3-60 5-2 10 F5-1 4.21 139M 1968 Philippines A 1 16 3 3-60 5-2 10 F5-1 —^(c) 120M 1967Pakistan A 1 16 56 3-60 5-2 10 F5-1  4.21 S5611 1977 Australia A 1 16 33-60 5-2 10 F5-1 — 106 1967 Morocco A 1 16 3 3-60 5-2 10 F5-1  4.21 3931968 Greece A 1 16 3 3-59 5-2 10 F5-1 — 322/85 1985 East Germany A 2 1652 3-80 5-2 10 F5-2  4.21 79128 1979 China A 3 54 10 3-60 7-1 10 F5-5 —BZ 133 1977 Netherlands B 1 2 3 3-60 7 16 F5-1 NT^(d) 79126 1979 China A3 22 10 3-01 7-3 10-5 F5-5  4 ST-4 complex/subgroup IV A4/M1027 1937 USAA 4 16 45 3-26 5-2 10 F1-5  4.21 26 1963 Niger A 4 45 5 3-26 7 13 F1-5 —243 1966 Cameroon A 4 45 5 3-26 7 13 F1-5 — 2059001 1990 Mali A 4 45 53-46 7 13 F1-5  4.21 10 1963 Burkina Faso A 4 21 5 3-26 7 13-1 F1-5 —255 1966 Burkina Faso A 4 24 5 3-26 7-5 13-1 F1-5  4.21 S3131 1973 GhanaA 4 21 5 3-26 7 13-1 F1-5 — 690 1980 India A 4 21 5 3-26 7 13-1 F1-5 4.21 C751 1983 Gambia A 4 21 5 3-26 7 13-1 F1-5 — 1014 1985 Sudan A 421 5 3-26 7 13-1 F1-5 — D8 1990 Mali A 4 21 5 3-26 7 13-1 F1-5 — ST-5complex/subgroup III IAL2229 1976 Brazil A 5 19 55 3-27 20  9 F2-1 — 1531966 China A 5 19 7 3-47 20  9 F3-1  4.21 154 1966 China A 6 19 7 3-4720  9 F3-1  4.21 14/1455 1970 USSR A 5 19 7 3-47 20  9 F3-1  4.21 S43551974 Denmark A 5 20 7 3-27 5-1 9 F3-1  4.21 7891 1975 Finland A 5 19 73-27 20  9 F3-1  4.21 F4698 1987 Saudi A 5 19 11 3-47 20  9 F3-1 — H19641987 UK A 5 19 11 3-47 20  9 F3-1 — F6124 1988 Chad A 5 19 11 3-47 20  9F3-1 — 92001 1992 China A 7 19 7 3-62 20  9 F3-1 — 11-004 1984 China A 519 54 3-61 20  9 F3-8 — 80049 1963 China A 5 16 5 3-56 5-2 10 F1-5  4ST-8 complex/cluster A4 BZ 10 1967 Netherlands B 8 7 4 2-20 5-1  2-2F3-9  2b B6116/77 1977 Iceland B 10 7 17 2-03 5-1  2-2 F1-4 — BZ 1631979 Netherlands B 9 1 38 2-03 21  16 F1-7  2b G2136 1986 England B 8 4620 2-03 5-2 10-1 F3-6 — SB25 1990 South Africa C 8 51 4 2-03 18-1  3F3-9 — AK22 1992 Greece B 8 16 4 2-30 5-2 10 F3-9 — 94/155 1994 NewZealand C 66 9 4 2-21 5 2 F3-9 312 901 1996 England C 8 9 18 2-03 5 2F1-7 — ST-11 complex/ET-37 38VI 1964 USA B 11 9 47 2-02 5 2 F1-1 —complex NG P20 1969 Norway B 11 9 9 2-19 5 2 F1-1  2a F1576 1984 Ghana C11 9 9 2-47 5 2 F1-1  2a 500 1984 Italy C 11 9 9 2-02 5 2 F1-1  2aMA-5756 1985 Spain C 11 27 6 2-02 5  2-1 F5-5  2a M597 1988 Israel C 1127 6 2-02 5  2-1 F5-5  2a D1 1989 Mali C 11 27 37 2-02 5  2-1 F5-4  2a90/18311 1990 Scotland C 11 27 6 2-31 5  2-1 F5-5 NT L93/4286 1993England C 11 67 27 2-02 5-1 10-4 F3-6 — BRAZ10 1976 Brazil C 11 70 362-02 5-1 10-1 F1-10  2a ST-32 complex/ET-5 8680 1987 Chile B 32 41 153-24 7-2 3 F3-1 15 complex BZ 83 1984 Netherlands B 34 16 53 3-01 5-2 10F5-1 NT 204/92 1992 Cuba B 33 4 26 3-08 19  15 F5-1 — EG 329 1985 EastGermany B 32 60 49 3-24 7-1 16 F1-2 15 NG 080 1981 Norway B 32 2 1 3-247 16 F3-3 15 BZ 169 1985 Netherlands B 32 14 1 3-14 5-2 16 F3-3 NT 44/761976 Norway B 32 2 1 3-24 7 16 F3-3 — NG144/82 1982 Norway B 32 2 1 3-637 16 F3-3 15 196/87 1987 Norway C 32 61 1 3-24 7-2  16-12 F3-3 15 NGPB24 1985 Norway B 32 57 1 3-24 7-2 16-7 F3-3 NT ST-41/44 complex/931905 1993 Netherlands B 41 39 2 3-16 7-2 4 F1-5 — Lineage 3 50/94 1994Norway B 45 39 2 3-51 7-2 4 F1-5 — 88/03415 1988 Scotland B 46 39 2 3-497-2 4 F1-5 — 91/40 1991 New Zealand B 42 39 2 3-01 7-2 4 F1-5  4 AK501992 Greece B 41 39 16 3-52 7-2 4 F1-5 — BZ198 1986 Netherlands B 41 392 3-01 7-2 4 F1-5 NT M-101/93 1993 Iceland B 41 39 2 3-01 7-2 4 F1-5 —M40/94 1994 Chile B 41 39 2 3-53 7-2 4 F1-5 — N45/96 1996 Norway B 41 3916 3-01 7-2 4 F1-5 — 400 1991 Austria B 40 52 2 3-36 7-2 13-2 F1-5 — NGE30 1988 Norway B 44 1 46 3-45 21  16 F1-7  4 NG H15 1988 Norway B 43 652 3-54 19  15-2 F1-5  8 NG H36 1988 Norway B 47 18 41 3-16 5-1  2-2 F1-7 8 ^(a)PorA epitopes VR1 and VR2 together with the FetA VR weredetermined by translation of nucleotide sequences followed by databaseinterrogation. ^(b)The serotype of the PorB protein is stated, whereavailable. ^(c)—, serotype not available. ^(d)NT: not typable.

TABLE 2 Genetic and antigenic diversity among 78 hyper-invasivemeningococci. Nucleotide sequence Number of Proportion of Mean Number ofNumber of VR Locus length (bp) alleles segregating sites p-distancepeptide sequences combinations^(a) porA 1086-1149 33 0.14 0.041 33 26fetA 1977-2019 33 0.19 0.051 31 16 porB^(b)  927-1020 31 0.36 0.131 2826 porB2 1020 8 0.05 0.016 8 6 porB3 927-939 23 0.10 0.031 20 2^(a)porA, VR1 and VR2 combinations; fetA, VR sequences, porB, variableloops I, IV-VIII combinations. ^(b)Divergent, alternate alleles (porB2and porB3) are present at the meningococcal porB locus.

EXAMPLE 2 Vaccine Compositions Composition of Meningococcal PorA/FetAVaccines

The analysis and methodology presented in Example 1 allows design ofparticular vaccine compositions according to the present invention.

Table 3 shows seven protein-based anti-meningococcal vaccinecompositions that contain different combinations of the purified PorAand FetA proteins according to the present invention.

These ‘recipes’ are advantageously consistent with currently availableepidemiological information.

The number of proteins required in each vaccine is given, together withthe particular epitopes included and together with the coverage attainedfor a number of different scenarios.

The vaccine coverage has been calculated for a number of isolatecollections as the percentage of isolates with at least one epitopehomologous to a vaccine epitope. The collection of 107 isolates wasassembled in 1996 to be representative of global disease in the latterhalf of the twentieth century. The meningococci belonging to the UKisolate collections from 1975-1995 are representative of thedisease-associated isolates obtained in the UK over this period.

The African/Asian vaccine particularly high coverage given therelatively limited diversity of disease-causing meningococci in theseareas.

For comparative purposes, the coverage of each of these isolatecollections that is attained by the prior art meningococcal serogroup CConjugate (MCC) vaccine, introduced on a population scale in 1999, isgiven as a comparison. It is noteworthy that this vaccine, currently inroutine use, is only approximately one-third to one-half as effective aseach of the three clonal complex directed PorA/FetA vaccine compositionsaccording to the present invention (ST-11, ST-32 and ST-41/44).Furthermore, it is only one-sixth to one-ninth as effective as thestandard/enhanced and geographically directed vaccine compostionsaccording to the present invention (see first four rows of Table 3),based on the percentage coverage of the global collection of 107disease-causing isolates. Thus the compositions of the present inventionare extremely effective and considerably better than prior artcompositions such as the MCC vaccine.

A summary of the compositions given in table 3 is presented below:

Standard P1.5-1 P1.2-2 F1-5 P1.5-2 P1.16 F3-1 P1.5 P1.10 F5-1 P1.7P1.13-1 F3-9 P1.7-2 P1.4 F5-5 P1-20 P1.9 Enhanced P1.5-1 P1.2-2 F1-5 P1,5-2 P1.16 F3-1 P1.5 P1.10 F5-1 P1,7 P1.13-1 F3-9 P1.7-2 P1.4 F5-5 P1-20P1.9 P1.19 P1.15 Europe/USA P1.5-1 P1.2-2 F1-5 P1.5-2 P1.16 F3-6 P1.5P1.10 F5-1 P1.7 P1.4 F4-1 P1.7-2 P1.2 F3-9 P1.19 P1.15 F1-7 Africa/AsiaP1.5-2 P1.10 F5-1 P1.7 P1.13-1 F1-5 P1-20 P1.9 F3-1 ST-11 Complex P1.5P1.2 F3-6 F5-1 ST-32 Complex P1.7 P1.16 F3-1 P1.19 P1.15 ST-41/44Complex P1.7-2 P1.4 F1-5 F1-7

TABLE 3 Table: Recipes for PorA/FetA meningococcal vaccines. Epitopecomposition Number % coverage of isolate collection PorA PorA of 1071975 1985 1995 1975-1995 Vaccine VR1 VR2 FetA proteins global UK UK UKUK Standard P1.5-1 P1.2-2 F1-5 11 88.79 84.80 90.00 95.00 89.54 P1.5-2P1.16 F3-1 P1.5 P1.10 F5-1 P1.7 P1.13-1 F3-9 P1.7-2 P1.4 F5-5 P1-20 P1.9Enhanced P1.5-1 P1.2-2 F1-5 12 90.65 88.80 91.00 96.00 91.69 P1.5-2P1.16 F3-1 P1.5 P1.10 F5-1 P1.7 P1.13-1 F3-9 P1.7-2 P1.4 F5-5 P1-20 P1.9P1.19 P1.15 Europe/ P1.5-1 P1.2-2 F1-5 12 81.31 92.80 90.00 94.00 92.31USA P1.5-2 P1.16 F3-6 P1.5 P1.10 F5-1 P1.7 P1.4 F4-1 P1.7-2 P1.2 F3-9P1.19 P1.15 F1-7 Africa/ P1.5-2 P1.10 F5-1 6 60.74 34.40 66.00 51.0049.23 Asia P1.7 P1.13-1 F1-5 P1-20 P1.9 F3-1 ST-11 P1.5 P1.2 F3-6 323.36 47.20 29.00 43.00 35.80 complex F5-1 ST-32 P1.7 P1.16 F3-1 3 30.8412.80 42.00 17.00 23.08 complex P1.19 P1.15 ST-41/44 P1.7-2 P1.4 F1-5 332.71 24.80 33.00 42.00 32.62 complex F1-7 MCC (for N/A N/A N/A N/A11.00 20.00 25.00 36.00 26.46 comparison)

EXAMPLE 3 Cloning and Expression of PorA/FetA Epitopes

Expression vector pET30 EkLIC vector (Novagen) is used in conjunctionwith ligation-independent cloning (LIC), which advantageously requiresno restriction digests or ligation. This example illustrates thepotential for co-expression of multiple proteins.

The expression system chosen in this example has the features of beingIPTG inducible from T7 lac promoter. The system incorporates a His-tagfor column purification (majority of clones with N-terminal His-tag,some with C-terminal His tag also). The system incorporates anenterokinase site for cleaving off N-terminal His tag if necessary ordesirable. The system advantageously produces inclusion bodies for easeof purification—PorA and FetA genes are cloned without signal sequenceto allow their expression in inclusion bodies.

In this example, the pET 30 EkLIC vector is used with a cloning methodcomprising PCR-amplifying FetA and PorA genes with specific endscompatible with LIC cloning. These are then treated with T4 DNApolymerase and dATP to create 5′ overhangs. The treated insert is thenannealed to pre-linearised vector. The annealed constructs are thentransformed into a cloning host leading to formation of circularplasmid. These plasmids are then screened by transforming into anexpression host. Other methods such as sequencing, western blots (PorAMAbs, His-tag MAb) are employed to verify the clones as required.

PorA types cloned are shown in table A; * Strain 3072 from the enhancedcombination, all other strains make up the standard combination.

FetA types cloned are shown in table B; All these types are for thestandard vaccine combination

EXAMPLE 4 Production of PorA/FetA Protein Antigens

In this example, IPTG induction is used to trigger expression of clonedPorA/FetA protein antigens from example 3. BL21 (DE3) transformed withthe PorA/FetA clones are grown on Luria agar/broth supplemented with 30μg/ml Kanamycin. Basal expression is suppressed by inclusion of 1%glucose.

Protocol: Production

Starter culture—Inoculate 50 ml LB in universal with one colony BL21(DE3) clone and grow overnight at 37° C. in shaking incubator. MeasureOD600 and inoculate 500 ml LB to OD600=0.05. Incubate at 37° C. inshaking incubator, 250 rpm to OD600=0.5-0.6. Induce by adding IPTG to 1mM final concentration. Return to shaking incubator for 2-3 hours.Transfer culture to centrifuge tubes. Incubate on ice for 5 mins.Harvest cells by centrifugation and freeze at −70° C.

Protocol: Inclusion Body Purification

Resuspend the cell pellet in BugBuster™ protein extraction reagent. AddLysonase (Lysozyme and nuclease). Incubate on a shaking platform untilno longer viscous. Spin 12.5 k rpm 20 min at 4° C. Resuspend the pelletin the same volume of BugBuster™ reagent. Add rLysozyme solution. Vortexand incubate at room temperature for 5 min. Add 6 vol 1:10 dilutedBugBuster™ reagent. Spin 6.5 k rpm 15 min at 4° C. to collect theinclusion bodies.

Protocol: Further Protein Purification from Inclusion Bodies

Wash inclusion bodies with 0.5 vol of 1:10 diluted BugBuster™. Repeatwash, spin 12.5 k rpm for 15 min at 4° C. and remove the supernatant.Denature pellet to 20 mg wet weight/ml in TE 8.0 M urea. Spin 12.5 krpm, 10 mins. Make up Supernatant to 4.0 M urea, 0.5 M NaCl, 1% Z3-14.Refold by dialysing against 20 mM Tris HCl pH7.9, 250 mM NaCl, 0.05%Z3-14. Spin 10 K, 10 mins. Reserve the supernatant containingsolubilised protein.

Following inclusion body purification, it is preferred to proceed with atag (in this example 6his tag) based purification—in this example Nickelcolumn purification is used.

Nickel Column Purification

His-bind Resin (Novagen) is used. Completely resuspend the resin.Transfer the slurry (2× column volume) to a column. Allow the resin topack under gravity flow.

Charge and equilibrate the column by applying:3 volumes sterile deionised water5 volumes 1×Charge Buffer (Nickel sulphate)3 volumes 1×Binding Buffer+0.05% Z3-14

Protocol: Column Chromatography

Filter protein sample through 0.45 urn filter. Bind protein extract tocolumn.Wash with 10×vol. Binding Buffer+0.05% Z3-14 (20 mM Imidazole)Wash with 6×vol. Wash Buffer+0.05% Z3-14 (60 mM Imidazole)Elute with 6×vol. Elute Buffer+0.05% Z3-14 (1M Imidazole)Final buffer: Dialyse against 10 mM Tris HCl, 150 mM NaCl, 0.1%TritonX-100.

In this example, PorA P1.7-2,4 are expressed. Referring to FIG. 2, lane1 is molecular weight markers, numbers are in kDa; lanes 2-6 areun-induced 1-5 hours; lanes 7-11 are 1 mM IPTG treatment 1-5 hours.

Referring to FIG. 3, PorA P1.7-2,4 fractionation is shown. Lane 0 ismolecular weight markers, numbers are in kDa; lane 1 is Whole cell t=0;lane 2 is supernatent IB extraction; lane 3 is Wash 2 from IBextraction; lane 4 is Wash 3 from IB extraction; lane 5 is Insolublefraction after denaturation; lane 6 is Soluble fraction afterdenaturation; lane 7 is H is column Elution fraction 1; lane 8 is H iscolumn Elution fraction 2.

According to this example, approx. 6 mg purified protein is producedfrom 500 ml culture after inclusion body (IB) extraction and His-bind(Nickel column) purification.

TABLE A Por A Types cloned Strain for PorA PorA VR1 (MAb) VR2 (MAb) 2726P1.7- PLPNIQPQVTKR YYTKDTNNNLTLV 2341 P1.5,10 PLQNIQPQVTKR HFVQNKQNQRPTL2763 P1.7,13- AQAANGGASGQVKVT YWTTVNTGSATTT 2757 P1.7-2,4AQAANGGASGQVKVT HVVVNNKVATHVP 2760 P1.20,9 QPQTANTQQGGKVKV YVDEQSKYHA2794 P1.5-1,2- PLQNIQQPQVTKR HFVQQTPQSQPTLV 3072* P1.19,15PPSKSQPQVKVTKA HYTRQNNADVFVP 2828 P15-2,10 PLPNIQPQVTKR HFVQNKQNQRPTL2740 P1.5,2 PLQNIQPQVTKR HFVQQTPKSQPTLV

TABLE B FetA types cloned Strain for FetA fetA VR 2757 F1-5SQFKIEDKEKATDEEKNKNRENEKIAKAYRLT 2760 F3-1GEFSIPTKEKKNGKEVDKPMEQQKKDRADEATVHAYKLS 2769 F5-1GEFEISGKKKDPKDPKKEIDKTDEEKAKDKKDMDLVHSY KLS 2758 F3-9SKFSIPTTEKKNGQDVAKPADQQAKDRKDEALVHSYRLT 2786 F5-5GKFKISDKKPDPNDPTKEIDKDAAEKAKDKKDMDLVHSY KLS

1. A composition comprising at least one purified PorA protein antigenand at least one purified FetA protein antigen.
 2. The compositionaccording to claim 1 wherein said PorA/FetA antigens are antigenicallyvariable antigens comprising the variable regions of PorA/FetA.
 3. Thecomposition according to claim 1 wherein the composition comprises atleast one PorA VR1 epitope and at least one PorA VR2 epitope, whereinsaid epitopes are selected from the list presented in Table
 3. 4. Thecomposition according to claim 1 wherein the composition comprises atleast one FetA epitope selected from the list presented in Table
 3. 5.The composition according to claim 1 wherein the composition comprisesPorA epitopes P1.5-2, P1.10, P1.7 and P1.13-1 and FetA epitopes F5-1 andF1-5.
 6. The composition according to claim 5 wherein the compositionfurther comprises PorA epitopes P1-20 and P1.9, and FetA epitope F3-1.7. The composition according to claim 5 wherein the compositioncomprises PorA epitopes P1.5-1, P1.2-2, P1.5-2, P1.16, P1.5, P1.10,P1.7, P1.7-2 and P1.4, and FetA epitopes F1-5, F5-1 and F3-9.
 8. Thecomposition according to claim 7 wherein the composition furthercomprises PorA epitopes P1.13-1, P1-20 and P1.9, and FetA epitopes F3-1and F5-5.
 9. The composition according to claim 8 wherein thecomposition further comprises PorA epitopes P1.19 and P1.15.
 10. Thecomposition according to claim 7 wherein the composition furthercomprises PorA epitopes P1.2, P1,19 and P1.15, and FetA epitope F1-7.11. The composition according to claim 1 wherein the compositioncomprises PorA epitopes P1.5 and P1.2, and FetA epitopes F3-6 and F5-1.12. The composition according to claim 1 wherein the compositioncomprises PorA epitopes P1.7, P1.16, P1.19 and P1.15, and FetA epitopeF3-1.
 13. The composition according to claim 1 wherein the compositioncomprises PorA epitopes P1.7-2 and P1.4, and FetA epitopes F1-5 andF1-7.
 14. The composition according to claim 1 further comprising one ormore components selected from the group consisting of transferrinbinding proteins, PorB, Opa, and NspA.
 15. The composition according toclaim 14 wherein said further component is selected from the groupconsisting of transferrin binding protein, PorB and Opa.
 16. Thecomposition according to claim 15 wherein said further component is Opa.17. A method of immunizing a subject against Neisseria meningitidisinfection comprising administering to said subject an effective amountof a vaccine composition according to claim
 1. 18. A method of inducingan immune response against Neisseria meningitidis infection comprisingadministering to said subject an effective amount of a vaccinecomposition according to claim
 1. 19. A method of producing a vaccinecomposition against an organism comprising the steps of: (i) providingsurface protein sequences for said organism; (ii) selecting from saidsurface proteins those which are antigenically variable; (iii)determining the incidence of each of said antigenically variableproteins in clinical occurrences of said organism; (iv) selecting asub-group of antigens from said antigenically variable surface proteinsto provide optimal representation of different isolate(s) of theorganism whilst including the minimum number of individual antigens; and(v) providing the antigens selected in step four in a vaccinecomposition.
 20. The method according to claim 19 wherein the individualantigens for inclusion in the composition are further selected asinducing equivalent immune responses.
 21. The method according to claim19 wherein said antigens are further selected to be representative ofvariation between multiple disease associated strains of said organism.22. A composition comprising a combination of PorA and FetA epitopes asset out in Table
 3. 23. A purified protein composition comprising acombination of PorA and FetA epitopes selected from the group consistingof P1.2, P1.2-2, P1.4, P1.5, P1.5-1, P1.5-2, P1.7, P1.7-2, P1.9, P1.10,P1.16, P1.13-1, P1.15, P1.19, and P1-20 and FetA epitopes F1-5, F1-7,F3-1, F3-6, F3-9, F5-1, and F5-5.
 24. The composition according to claim1 which is a vaccine composition.
 25. The composition according to claim24 wherein said composition is an outer membrane vesicle vaccine. 26.The method of claim 19, wherein a predetermined number of antigens isincluded in said vaccine composition. 27, The method of claim 19,wherein step (iv) is used to select a sub-group of antigens from saidantigenically variable surface proteins to provide maximalrepresentation of different isolate(s) of the organism while including apredetermined number of individual antigens.
 28. The method of claim 19,wherein the coverage of the composition is predetermined to ageographical spread of isolates.
 29. The method of claim 19, wherein thecoverage of the composition is predetermined to a particular clone orcollection of isolates.
 30. The method of claim 19, wherein step (iv) isused to select a sub-group of antigens from said antigenically variablesurface proteins to provide a desired coverage by choosing a singleantigen that provides the greatest possible increase in coverage andadding this to the sub-group of selected antigens until addition of afurther antigen would not further increase the coverage of isolate(s) ofthe organism.
 31. The method of claim 19, wherein the subgroup ofantigens of step (iv) includes multiple antigens present on a singleisolate of said organism.
 32. The method of claim 19, wherein thesubgroup of antigens of step (iv) includes only antigens each of whichis present on a separate isolate of said organism.
 33. The method ofclaim 19, wherein the organism is Neisseria meningitidis.